The Profile of Post-translational Modifications of Histone H1 in Chromatin of Mouse Embryonic Stem Cells.

Linker histone H1 is one of the main chromatin proteins which plays an important role in organizing eukaryotic DNA into a compact structure. There is data indicating that cell type-specific post-translational modifications of H1 modulate chromatin activity. Here, we compared histone H1 variants from NIH/3T3, mouse embryonic fibroblasts (MEFs), and mouse embryonic stem (ES) cells using matrix-assisted laser desorption/ ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS). We found significant differences in the nature and positions of the post-translational modifications (PTMs) of H1.3-H1.5 variants in ES cells compared to differentiated cells. For instance, methylation of K75 in the H1.2-1.4 variants; methylation of K108, K148, K151, K152 K154, K155, K160, K161, K179, and K185 in H1.1, as well as of K168 in H1.2; phosphorylation of S129, T146, T149, S159, S163, and S180 in H1.1, T180 in H1.2, and T155 in H1.3 were identified exclusively in ES cells. The H1.0 and H1.2 variants in ES cells were characterized by an enhanced acetylation and overall reduced expression levels. Most of the acetylation sites of the H1.0 and H1.2 variants from ES cells were located within their C-terminal tails known to be involved in the stabilization of the condensed chromatin. These data may be used for further studies aimed at analyzing the functional role played by the revealed histone H1 PTMs in the self-renewal and differentiation of pluripotent stem cells.

Post-translational modifications of linker histone H1 variants in mammals.

The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2-H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

INTERMOLECULAR INTERACTIONS IN THE SOLUTIONS OF SERUM ALBUMIN.

The mechanisms of intermolecular protein complex formation were studied by the example of monomers, oligomers and aggregates of bovine serum albumin (BSA) depending on the protein concentration, pH and urea concentration. Using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and PAG electrophoresis, we have shown that there is dynamic equilibrium between monomers and aggregates in BSA solution. Decreasing pH of the solution (4.0­1.0) resulted in increasing sizes of the aggregates. In the solutions with low urea concentrations (below 2 M), the sizes of aggregates decreased, while higher urea concentrations induced formation of larger aggregates due to the unfolding of the protein.

[Analysis of the secondary structure of linker histone H1 based on IR absorption spectra].

This work was carried out to compare the effectiveness of the methods of infrared spectroscopy in the amide I region and UV circular dichroism to the analyze the protein secondary structure by the example of linker histone H1 and bovine serum albumin (BSA). It has been shown that the application of a diamond ATR cell quantifies the proportion of α-helices and ß-structures in a good agreement with UV circular dichroism spectroscopy. It has been shown that histone H1 is able to aggregate, which results in considerable changes in its secondary structure.

[The structure of the complexes of DNA with nonhistone chromosomal protein HMGB1 and the histone H1 in the presence of manganese ions. II. Vibrational circular dichroism spectroscopy].

Complexes of DNA with nonhistone chromosomal protein HMGB1 and histone H1 in the presence of manganese ions were studied using absorption and circular dichroism spectroscopy in infrared region. It was shown that the approach provides good results for solutions containing large particles, which cause light scattering in UV region. It was also shown that the manganese ions are able to coordinate to the chemical groups of DNA as well as to the carboxylic amino acid residues of the protein HMGB1. The latter stimulates DNA condensation and slightly weakens DNA-protein interactions in the complex.

[Investigation of transcriptional regulation of Oct4 (POU5F1) gene with distal enhancer].

Investigations of transcriptional regulation of Oct4 gene in mouse embryonic stem cells have revealed an important cis-element--the distal enhancer (DE). DE consists of two functionally significant elements--DEa and DEb. Both elements are necessary to complete the DE-mediated expression of Oct4 gene in pluripotent cells. The most likely candidates for the binding site DEb are Oct4 itself in complex with Sox2 protein. It remains unclear which transcriptional proteins bind to the DEa site and what is the mechanism of the co-operation between the DEa and the DEb. Through the use of using the EMSA and chromatographic fractionation of proteins from extracts of mouse embryonic stem cells and mouse tissues, were isolated proteins specifically interacting with the sequence DEa Oct4 gene.

[The structure of the complexes of DNA with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions. I. Circular dicroism spectroscopy].

The mechanisms of interaction of the non-histone chromosomal protein HMGB1 and linker histone H1 with DNA have been studied using circular dichroism and absorption spectroscopy. Both of the proteins are located in the inter-nucleosomal regions of chromatin. It was demonstrated that properties of the DNA-protein complexes depend on the protein content and can not be considered as a simple summing up of the effects of individual protein components. Interaction of HMGB1 and H1 proteins is shown to be co-operative rather than competitive. Lysine-rich histone H1 facilitates the binding of the HMGB1 with DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino-acid residues in the C-terminal domain of the HMGB1 protein. The observed joint action of the and H1 proteins stimulates DNA condensation with formation of the anisotropic DNA-protein complexes with typical psi-type CD spectra. Structural organization of the complexes depends not only on the DNA-protein interactions, but also on the interaction between HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the character of interactions between the components in the triple DNA-HMGB1-H1 complex. Binding of Mn2+ ions causes the weakening of the DNA-protein interactions and strengthening the protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.

[Conformational peculiarities of nuclear protein HMGB1 and specificity of its interaction with DNA].

Changes in the secondary structure of DNA and non-histone chromosomal protein HMGB1 were studied by circular dichroism and UV spectroscopy. We have demonstrated that the HMGBI protein is able to change its secondary structure upon binding to DNA. We estimated the proportion of bound protein on the assumption that there were two spectrally distinguishable forms of the HMGB1 in solution. The bound protein fraction decreases with increasing protein to DNA ratios (r) from 0.48 at r = 0.13 to 0.06 at r = 2.43. It has been shown that HMGB1 is able to induce considerable changes in DNA structure even when the amount of the protein directly associated with DNA is low.

[Generation of rat induced pluripotent stem cells: the analysis of reprogramming and culturing media].

The rat represents very important, superior in many respects to the mous, animal model for studying pharmacology, physiology, ageing, cardiovascular etc. However, numerous attempts to derive rat ES cells necessary to carry out loss-of-gene-function studies have not been successful thus far. Therefore rat induct pluripotent stem cells (or riPS) should provide a notable alternative to ES cell, allowing to study gene functions in this valuable animal model. Here we report an improved lentivirus-based riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We show that the excision of proviruses does not affect neither karyotype and pluripotency state of these cells. Also, we propose genetic tool for an improvement of the quality of riPS cells in culture. These data may prompt further iPS-based gene targeting in rat as well as the development iPS-based gene therapies, using this animal model.

[The interaction of DNA with sperm-specific histones of the H1 family].

We have studied the interactions of DNA with sperm-specific histones of the H1 family of sea urchin Strongylocentrotus intermedius, sea starfish Aphelasterias japonica and bivalve mollusk Chlamis islandicus using circular dichroism and DNA melting analysis. It was shown that echinoderm's sperm H1 protein has additional alpha-helical domains in its C-terminus and it demonstrates stronger DNA compaction. The differential melting curves of DNA-protein complexes have two peaks. The low temperature peak characterized the melting temperature of free DNA within the complex. The higher temperature peak characterizes the melting temperature of DNA bond to protein. DNA is found to be in the most stable state in the complexes with mollusk sperm H1 protein.

[Nucleosome remodeling on the regulatory region of the rat tryptophan dioxygenase (tdo) gene during transcription in vivo].

Two DNase I-hypersensitive regions are identified on the regulatory region of the rat tryptophan dioxygenase (tdo) gene which is expressed tissue-specifically under control ofglucocorticoid hormones. DNase I-hypersensitive regions are identified in position -470 and in the vicinity of the first upstream gene. Micrococcal nuclease digestion pattern of this region shows disturbances in the regular cleavage and appearance of shorter DNA molecules than nucleosomal DNA. However the control experiments demonstrate that the same DNA region could be involved in the regular nucleosome core particles under in vitro reconstitution. Taken together, these data show that the nucleosome array in the regulatory region of the actively transcribed tdo gene in vivo is disturbed.

[E-cadherin as a novel surface marker of spermatogonial stem cells].

Spermatogenesis is a fundamental biological process that ensures gee transmission from one generation to another trough gametes. This process relies on a rare population of testicular cells, called spermatogonial stem cells (SSCs), that self-renew throughout adult male life and differentiate into mature gametes. Despite the longstanding research of SSCs, their biological properties remain largely unknown which is partly due to very limited availability of these cells. Here we show that cell adhesion protein E-cadherin is a highly specific surface marker of mouse SSCs, which can be successfully used for their enrichment.

[Transcription factors of the nuclear factor 1 (NF1) family. Role in chromatin remodelation].

Site-specific transcription factors of the nuclear factor 1 (NF1) family are ubiquitous, are encoded by four genes in mammals, and play a role in regulating transcription of approximately 100 cell and viral genes. The NF1 proteins are characterized by the ability to act as both transcription factors and viral replication factors and by a low affinity for nucleosomal DNA. The review considers the dual role of the NF1 family factors in hormone-mediated transactivation of mouse mammary tumor virus (MMTV) promoter: the factors are essential for both transcription activation and nucleosome remodeling. Emphasis is placed on the role of the NF1 family factors in the formation and maintenance of a preset chromatin structure in the MMTV promoter region and the regulatory elements of the lysozyme gene during macrophage differentiation. Since disruption of individual NF1 family genes caused pathological changes in human and animal tissues, the NF1 family proteins are subjects of basic and applied research.

[Structural organization of DNA complexes with proteins HMGB1 and HMGB1-(A+B)].

The interaction between DNA and the nonhistone proteins HMGB1 and HMGB1-(A+B) has been studied using circular dichroism and scanning force microscopy. The recombinant protein HMGB1-(A+B) has no negatively charged C-terminal domain characteristic for HMGB1. Our earlier suggestion about the structural interaction of tandem HMGB1-domains of the recombinant protein with DNA was confirmed. It was shown that the C-terminal part modulates the interactions of HMGB1-domains with DNA. Without the C-terminal sequence, the HMGB1-(A+B) protein forms DNA-protein complexes with the ordered supramolecular structure.

The effect of manganese(II) on the structure of DNA/HMGB1/H1 complexes: electronic and vibrational circular dichroism studies.

The interactions were studied of DNA with the nonhistone chromatin protein HMGB1 and histone H1 in the presence of manganese(II) ions at different protein to DNA and manganese to DNA phosphate ratios by using absorption and optical activity spectroscopy in the electronic [ultraviolet (UV) and electronic circular dichroism ECD)] and vibrational [infrared (IR) and vibrational circular dichroism (VCD)] regions. In the presence of Mn2+, the protein-DNA interactions differ from those without the ions and cause prominent DNA compaction and formation of large intermolecular complexes. At the same time, the presence of HMGB1 and H1 also changed the mode of interaction of Mn2+ with DNA, which now takes place mostly in the major groove of DNA involving N7(G), whereas interactions between Mn2+ and DNA phosphate groups are weakened by histone molecules. Considerable interactions were also detected of Mn2+ ions with aspartic and glutamic amino acid residues of the proteins.

[The structure of the complexes of DNA with non-histone chromosomal protein HMGB1 in the presence of manganese ions]. / Struktura kompleksov DNK s negistonovym khromosomnym belkom HMGB1 v prisutstvii ionov margantsa.

The complexes of DNA - HMGB1 protein - manganese ions have been studied using circular dichroism (CD) technique. It was shown that in such three-component system the interactions of both the protein and metal ions with DNA differ from those in two-component complexes. The manganese ions do not affect the CD spectrum of free HMGB1 protein. However, Mn2+ ions induce considerable changes in the CD spectrum of free DNA in the spectral range of 260-290 nm. The presence of Mn2+ ions prevents formation of the ordered supramolecular structures specific for the HMGB1-DNA complexes. The interaction of manganese ions with DNA has a marked influence on the local DNA structure changing the properties of protein-binding sites. This results in the serious decrease in cooperativity of the DNA-protein binding. Such changes in the mode of the DNA-protein interactions occur at concentrations as small as 0.01 mM Mn2+. Moreover, the changes in local DNA structure induced by manganese ions promote the appearance of new HMGB1 binding sites on the DNA double helix. At the same time interactions with HMGB1 protein induce alterations in the structure of the DNA double helix which increase with a growth of the protein/DNA ratio. These alterations make the DNA/protein complex especially sensitive to manganese ions. Under these conditions the Mn2+ ions strongly affect the DNA structure that reflects in abrupt changes of the CD spectra of DNA in the complex in the range of 260-290 nm. Thus, structural changes of the DNA double helix in the three-component DNA-HMGB1-Mn2+ complexes come as a result of the combined and interdependent interactions of DNA with Mn2+ ions and the molecules of HMGB1.

[The effect of Ca2+ ions on DNA compaction in the complex with non-histone chromosomal protein HMGB1]. / Vliianie ionov Ca2+ na kompaktizatsiiu DNK v komplekse s negistonovym khromosomnym belkov HMGB1.

The analysis of absorption and circular dichroism spectra in UV and IR regions showed that Ca2+ ions interact both with the phosphate groups of DNA and with the HMGB1 protein. Not only negatively charged C-terminal part of the protein molecule participates in interaction with metal ions but also its DNA-binding domains. The latter fact leads to the change of the mode of protein-DNA interaction. The presence of Ca2+ ions prevents formation of ordered supramolecular structures, specific for the HMGB1-DNA complexes, though promotes intermolecular aggregation. The structure of the complexes between DNA and the protein HMGB1 lacking C-terminal tail appears to be the most sensitive to the presence of Ca2+ ions. The data obtained allow to conclude that Ca2+ ions do not play a structural role in the HMGB1/DNA complexes and the presence of these ions is not necessary to DNA compaction in such systems.

The effect of manganese(II) on DNA structure: electronic and vibrational circular dichroism studies.

The interaction of DNA with Mn2+ was studied in absorbance and optical activity in the electronic and vibrational regions. Based on the data, several stages of the interaction were identified. Con formational transition towards the C-form of DNA was observed in solution at the molar ratio Mn2+/DNA-phosphates between 0.1 and 1.5. The exact ratio depended on the ionic strength and increased with increasing NaCl concentration. Although manganese interacted with the phosphates and bases of DNA at higher metal concentrations, it is unlikely that direct chelation occurred. A model for the interaction between manganese ions and DNA mediated by water is suggested destabilizing the double helix and partially breaking the hydrogen bonds between the base pairs. At high Mn2+ concentrations DNA aggregation was observed.

[HMG1 domains: the victims of circumstance]. / HMG1-domeny: zalozhniki obstoiatel'stv.

The method of circular dichroism (CD) was used to compare DNA behavior during its interaction with linker histone H1 and with non-histone chromosomal protein HMG1 at different ionic strength and at different protein content in the system. The role of negatively charged C-terminal fragment of HMG1 was analyzed using recombinant protein HMG1-(A + B), which lacks the C terminal amino acid sequence. The psi-type CD spectra were common for DNA interaction with histone H1, but no spectra of this type were observed in HMG1-DNA systems even at high ionic strength. The CD spectrum of the truncated recombinant protein at high salt concentration somewhat resembled the psi-type spectrum. Two very intense positive bands were located near 215 nm and near 273 nm, and the whole CD spectrum was positive. The role of C-terminal tail of HMG1 in formation of the ordered DNA-protein complexes is discussed.